Widespread efforts have been made in recent years to determine the sequence of the human genome, as well as the genomes of various other organisms. The advent of genomics has relied upon accurate and efficient DNA sequencing techniques, and the ability to determine the nucleotide sequence of a gene remains an essential component of molecular genetic research. The widespread use of DNA sequencing in biological research necessitates the development of new DNA sequencing techniques that are simpler and more efficient than traditional, commonly-used techniques.
Classical DNA sequencing techniques, such as the Sanger chain termination method (Sanger, F., et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977); incorporated herein by reference) and the Maxam and Gilbert chemical cleavage method (Maxam, A. M. and Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977); incorporated herein by reference), are somewhat cumbersome and inefficient, as both of these approaches require researchers to perform multiple reactions in order to derive a nucleotide sequence. Attempts to simplify DNA sequencing by coupling a single DNA amplification/synthesis reaction with sequence analysis (i.e., direct sequencing) have shown limited success, because these techniques often result in DNA damage and/or degradation (Das et al., Physiol Genomics 6: 57-80 (2001)), as well as low fidelity DNA synthesis (Lin et al., Biochemistry 40: 8749-8755 (2001); Xia et al., Proc Natl Acad Sci USA 99: 6597-6602 (2002); U.S. Pat. No. 5,939,292; U.S. Pat. No. 6,329,178; and U.S. Pat. No. 6,887,690).
Presently, there is a clear need to develop improved methods of sequencing DNA. More specifically, there is a need to develop reliable and efficient direct sequencing techniques that yield accurate DNA sequence information.